Transdiscal Administration Of Specific Inhibitors Of Pro-Inflammatory Cytokines

ABSTRACT

The present invention relates to injecting a high specificity cytokine antagonist into a diseased intervertebral disc.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/220,273, filed Mar. 20, 2014, which is a continuation of U.S.application Ser. No. 12/291,378, filed Nov. 7, 2008, now U.S. Pat. No.8,728,523, issued May 20, 2014, which is a continuation of U.S.application Ser. No. 11/881,926, filed Jul. 30, 2007, now Abandoned,which is a Divisional of U.S. application Ser. No. 10/456,948, filedJun. 6, 2003, now U.S. Pat. No. 7,344,716, issued Mar. 18, 2008, whichclaims the benefit of U.S. Provisional Application No. 60/470,098, filedMay 13, 2003. The entire teachings of the above applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

The natural intervertebral disc contains a jelly-like nucleus pulposussurrounded by a fibrous annulus fibrosus. Under an axial load, thenucleus pulposus compresses and radially transfers that load to theannulus fibrosus. The laminated nature of the annulus fibrosus providesit with a high tensile strength and so allows it to expand radially inresponse to this transferred load.

In a healthy intervertebral disc, cells within the nucleus pulposusproduce an extracellular matrix (ECM) containing a high percentage ofproteoglycans. These proteoglycans contained sulfated functional groupsthat retain water, thereby providing the nucleus pulposus within itscushioning qualities. These nucleus pulposus cells may also secretesmall amounts of cytokines as well as matrix metalloproteinases(“MMPs”). These cytokines and MMPs help regulate the metabolism of thenucleus pulposus cells.

In some instances of disc degeneration disease (DDD), gradualdegeneration of the intervetebral disc is caused by mechanicalinstabilities in other portions of the spine. In these instances,increased loads and pressures on the nucleus pulposus cause the cells toemit larger than normal amounts of the above-mentioned cytokines. Inother instances of DDD, genetic factors, such as programmed cell death,or apoptosis can also cause the cells within the nucleus pulposus toemit toxic amounts of these cytokines and MMPs. In some instances, thepumping action of the disc may malfunction (due to, for example, adecrease in the proteoglycan concentration within the nucleus pulposus),thereby retarding the flow of nutrients into the disc as well as theflow of waste products out of the disc. This reduced capacity toeliminate waste may result in the accumulation of high levels of toxins.

As DDD progresses, the toxic levels of the cytokines present in thenucleus pulposus begin to degrade the extracellular matrix (inparticular, the MMPs (under mediation by the cytokines) begin cleavingthe water-retaining portions of the proteoglycans, thereby reducing itswater-retaining capabilities). This degradation leads to a less flexiblenucleus pulposus, and so changes the load pattern within the disc,thereby possibly causing delamination of the annulus fibrosus. Thesechanges cause more mechanical instability, thereby causing the cells toemit even more cytokines, thereby upregulating MMPs. As this destructivecascade continues and DDD further progresses, the disc begins to bulge(“a herniated disc”), and then ultimately ruptures, causing the nucleuspulposus to contact the spinal cord and produce pain.

Olmarker, Spine 26(8), 2001, pp. 863-9(“Olmarker I”) and Aoki, Spine27(15), 2002, pp. 1614-17 teach that TNF-α appears to play a role inproducing the pain associated with the nucleus pulposus contacting nerveroots of the spinal cord.

US Published Patent Application No. US 2003/0039651 (“Olmarker II”)teaches a therapeutic treatment of nerve disorders comprisingadministration of a therapeutically effective dosage of at least twosubstances selected from the group consisting of TNF inhibitors (bothspecific and non-specific), IL-1 inhibitors, IL-6 inhibitors, IL-8inhibitors, FAS inhibitors, FAS ligand inhibitors, and IFN-gammainhibitors.

In the examples of Olmarker II, Olmarker II further teaches that thesesubstances are to be administered through systemic pathways. Inparticular, Olmarker II teaches that “the major contribution ofTNF-alpha may be derived from recruited, aggregated and maybe evenextravasated leukocytes, and that successful pharmacologic block may beachieved only by systemic treatment. [0133]. Of note, Olmarker IIappears to discourage the local addition of one therapeutic agent(doxycycline) to a transplanted nucleus pulposus. [0128].

PCT Published Patent Application No. WO 02/100387 (“Olmarker III”)teaches the prevention of neovasculariation and/or neo-innervation ofintervertebral discs by the administration of anti-angiogenicsubstances. Again, however, Olmarker III teaches systemic administrationof these therapeutic agents.

U.S. Pat. No. 6,419,944 (“Tobinick”) discloses treating herniated discswith cytokine antagonists, including infliximab. However, Tobinickteaches that local administration involves a subcutaneous injection nearthe spinal cord. Accordingly, Tobinick does not teach a procedureinvolving a sustained delivery of a drug for the treatment of DDD, nordirectly administering a specific cytokine antagonist (such asinfliximab) into the disc.

US Published Patent Application No. 2003/0049256 (Tobinick II) disclosesthat injection of such therapeutic molecules to the anatomic areaadjacent to the spine is accomplished by interspinous injection, andpreferably is accomplished by injection through the skin in the anatomicarea between two adjacent spinous processes of the vertebral column.

Tobinick II further teaches that TNF antagonists may be administered byinterspinous injection in the human and that the dosage level is in therange of 1 mg to 300 mg per dose, with dosage intervals as short as twodays. Tobinick II further discloses that Interleukin-1 antagonists areadministered in a therapeutically effective dose, which will generallybe 10 mg to 200 mg per dose, and their dosage interval will be as shortas once daily.

Tobinick, Swiss Med. Weekly, 2003, 133, 170-77 (“Tobinick III”) teachesboth perispinal and epidural administration of TNF inhibitors for spinerelated therapies.

Karppinen, Spine, 28(8), 203, pp. 750-4, teaches intravenously injectingor orally administering infliximab into patients suffering fromsciatica.

As with Tobinick I and II, Karppinen does not teach a procedureinvolving a sustained delivery of a drug for the treatment of DDD, nordirectly administering a specific cytokine antagonist (such asinfliximab) into the disc.

U.S. Pat. No. 6,352,557 (Ferree) teaches adding therapeutic substancessuch as anti-inflammatory medications to morselized extra-cellularmatrix, and injecting that combination into an interverterbral disc.

However many anti-inflammatory agents are non-specific and therefore mayproduce unwanted side effects upon other cells, proteins and tissue. Inaddition, the pain-reducing effect of these agents is typically onlytemporary. Lastly, these agents typically only relieve pain, and areneither curative nor restorative.

Alini, Eur. Spine J. 11(Supp.2), 2002, pp. S215-220, teaches therapiesfor early stage DDD, including injection of inhibitors of proteolyticenzymes or biological factors that stimulate cell metabolic activity(i.e., growth factors) in order to slow down the degenerative process.Alini I does not disclose inhibiting growth factors.

US Published Patent Application US 2002/0026244 (“Trieu”) discloses anintervertebral disc nucleus comprising a hydrogel that may deliverdesired pharmacological agents. Trieu teaches that these pharmacologicalagents may include growth factors such as TGF-B and anti-inflammatorydrugs, including steroids. Trieu further teaches that thesepharmacological agents may be dispersed within the hydrogel having anappropriate level of porosity to release the pharmacological agent at adesired rate. Trieu teaches that these agents may be released uponcyclic loading or upon resorption.

Takegami, Spine, 27(12), 2002, 1318-25 teaches that injecting TGF-B intothe disc space results in enhanced replenishment of the extracellularmatrix damaged by cytokines. Takegami further teaches that the half-lifeof a growth factor injected into the interveterbal disc can be expectedto be longer than that injected into a synovial joint because thenucleus pulposus is surrounded by the fibrous structure of the annulusfibrosus, thus providing a confined environment. Diwan, TissueEngineering in Orthopedic Surgery, 31(3) July 2000, pp. 453-464, reportson another Takegami paper that concluded that a delivery system allowingprolonged delivery (>3 days) would have to be used to obtain theobserved effect of the growth factor.

Alini, Spine 2003 28(5), pp. 446-54, discloses a cell seededcollagen-hyaluronan scaffold supplemented with growth factors such asTGF-B, bFGF, and IGF-1 for use in regenerating a nucleus pulposus.

Maeda et al. Spine 2000, 25(20 pp. 166-169, 2000 reports on the in vitroresponse to interleukin-1 receptor antagonist protein (IRAP) of rabbitannulus fibrosus exposed to IL-1. Maeda suggests that TRAP could beuseful in inhibiting the degradation of the disc.

Yabuki, Spine, 2001, 26(8), 870-5, teaches the use of an anti-TNF drugfor the treatment of sciatica.

U.S. Pat. No. 6,277,969 (“Le”) discloses the use of anti-TNF antibodiesfor therapy of TNF-mediated pathologies. Le teaches parentaladministration of the antibodies.

In sum, when investigators suggest the administration of specific TNF-ainhibitors or specific interleukin inhibitors, the investigators appearnot only to teach only the administration of those therapeutics totissue outside the disc, but it also appears to discourage thetrans-discal administration of therapeutic substances.

SUMMARY OF THE INVENTION

The present inventors have developed a number of procedures forefficaciously treating degenerative disc disease by drug therapy.

The present inventors have noted that although Tobinick, Olmarker andKarppinenen taught the therapeutic use of pro-inflammatorycytokine-antagonist monoclonal antibodies in treating sciatica, each ofthese investigators targeted tissue outside of the disc.

In accordance with the present invention, the present inventors havedeveloped a method of treating an intervertebral disc in which a highspecificity inhibitor of a pro-inflammatory cytokine is administeredtransdiscally (i.e., the target tissue is a degenerating disc).

There are believed to be several advantages to directly administeringthese therapeutic inhibitors to a targeted disc over the treatmentsdisclosed by Tobinick and Karppinenen:

First, since it is known that many cytokines (such as interleukins andTNF-α) also play roles in mediating the degradation of the extracellularmatrix (ECM) of the nucleus pulposus, injecting an antagonist orinhibitor of these proteins directly into the disc prevents the targetcytokine from inducing any further ECM degradation. In effect, thetransdiscal administration of the cytokine antagonist arrests the agingprocess of the degenerating disc. Accordingly, the present inventionseeks to treat the degenerative disc at a much earlier stage of DDD thanTobinick and Karppinenen and thereby prevents degradation of the ECM.

Second, it is further known that nerve ending nociceptors are presentwithin the annulus fibrosus, and that cytokines such as TNF irritatenerves. It is believed that injecting an anti-TNF antagonist into thedisc space also prevents the TNF from causing nerve irritation withinthe disc. Thus, the pain attributed to irritation of these nerves can beefficiently eliminated.

Third, since the annulus fibrosus portion of the disc comprises arelatively dense fibrosus structure, this outer component of the discmay provide a suitable depot for the high specificity cytokineantagonist (HSCA), thereby increasing its half-life in the disc.

Fourth, since the high specificity antagonist inhibits only the specificcytokine of interest, the HSCA may be combined with other therapeuticagents (such as TGF-B, or mesenchymal stem cells) that can also beinjected into the disc without reducing the effectiveness of thoseagents.

Fifth, since it is believed that many of the problematic cytokines areactually secreted by either nucleus pulposus or annulus fibrosus cells,transdiscal injection of the high specificity antagonists willadvantageously attack the problematic cytokines at their source oforigination.

Accordingly, in a first aspect of the present invention, there isprovided a method of treating an intervertebral disc having a nucleuspulposus, comprising the steps of:

a) transdiscally administering a formulation comprising a highspecificity cytokine antagonist (HSCA) into an intervertebral disc.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

For the purposes of the present invention, the terms “inhibitor” andantagonist” are used interchangeably. A protein may be inhibited at thesynthesis level, at the translation level, by shedding, by antibodies,or by soluble receptors. The term “patient” refers to a human having adegenerating disc.

For the purposes of the present invention “Transdiscal administration”includes, but is not limited to:

-   -   a) injecting a formulation into the nucleus pulposus of a        degenerating disc, preferably a relatively intact degenerating        disc,    -   b) injecting a formulation into the annulus fibrosus of a        degenerating disc, preferably relatively intact degenerating        disc.    -   c) providing the formulation in a patch attached to the outer        wall of the annulus fibrosus,    -   d) providing the formulation in a depot at a location outside        but closely closely adjacent the outer wall of the annulus        fibrosus (hereinafter, “trans-annular administration”.    -   e) providing the formulation in a depot at a location outside        but closely adjacent the endplates of the adjacent vertebral        bodies (hereinafter, “trans-endplate administration”.

Because DDD is a continuous process, the degenerating disc to which thetherapeutic drug is administered may be in any one of a number ofdegenerative states. Accordingly, the degenerating disc may be an intactdisc. The degenerating disc may be a herniated disc (wherein a portionof the annulus fibrosus has a bulge). The degenerating disc may be aruptured disc (i.e., wherein the annulus fibrosus has ruptured and bulknucleus pulposus has exuded). The degenerating disc may be delaminated(wherein adjacent layers of the annulus fibrosus have separated). Thedegenerating disc may have fissures (wherein the annulus fibrosus hasfine cracks or tears through which selected molecules from the nucleuspulposus can leak).

The present invention is directed to providing directly through adiseased intervertebral disc at least one highly specific cytokineantagonist capable of specifically inhibiting a cytokine (preferably, apro-inflammatory cytokine) present in the disc. Preferably, the HSCAinhibits the action of a specific pro-inflammatory cytokine released bydisc cells or by invading macrophages during the degenerative process.

In some embodiments, the antagonist is capable of specificallyinhibiting a pro-inflammatory cytokine selected from the groupconsisting of TNF-α, an interleukin (preferably, IL-1, I1-6 and IL-8) ,phospholipase A2 (PLA2), FAS, an FAS ligand, and IFN-gamma. Suchspecific inhibitors include those identified on pages 5-18 of OlmarkerII, the specification of which is incorporated by reference in itsentirety.

In some embodiments, the HSCA inhibits the cytokine by preventing itsproduction. In some embodiments, the HSCA inhibits the cytokine bybinding to a membrane-bound cytokine. In others, the HSCA inhibits thecytokine by binding to a solubilized cytokine. In some embodiments, theHSCA inhibitor inhibits the cytokine by both binding to membrane boundcytokines and to solubilized cytokine. In some embodiments, the HSCA isa monoclonal antibody (“mAb”). The use of mAbs is highly desirable sincethey bind specifically to a certain target protein and to no otherproteins. In some embodiments, the HSCA inhibits the cytokine by bindingto a natural receptor of the target cytokine.

In some embodiments, the HSCA inhibits the cytokine by preventing itsproduction. One example thereof is an inhibitor of p38 MAP kinase. Insome embodiments, the TNF inhibitor inhibits the TNF by binding tomembrane bound TNF in order to prevent its release from membrane. Inothers, the TNF inhibitor inhibits the TNF by binding to solubilizedTNF. One example thereof is etanercept. In some embodiments, the TNFinhibitor inhibits the TNF by both binding to membrane bound TNF and tosolubilized TNF. One example thereof is infliximab. In some embodiments,the HSCA inhibits the cytokine by binding to a natural receptor of thetarget cytokine.

Preferred TNF antagonists include, but are not limited to the following:etanercept (Enbrel.®.-Amgen); infliximab (Remicade.®.-Johnson andJohnson); D2E7, a human anti-TNF monoclonal antibody (KnollPharmaceuticals, Abbott Laboratories); CDP 571 (a humanized anti-TNFIgG4 antibody); CDP 870 (an anti-TNF alpha humanized monoclonal antibodyfragment), both from Celltech; soluble TNF receptor Type I (Amgen);pegylated soluble TNF receptor Type I (PEGs TNF-R1) (Amgen); andonercept, a recombinant TNF binding protein (r-TBP-1) (Serono).

TNF antagonists suitable for compositions, combination therapy,co-administration, devices and/or methods of the present invention(further comprising at least one anti body, specified portion andvariant thereof, of the present invention), include, but are not limitedto, anti-TNF antibodies (e.g., at least one TNF antagonist (e.g., butnot limited to a TNF chemical or protein antagonist, TNF monoclonal orpolyclonal antibody or fragment, a soluble TNF receptor (e.g., p55, p70or p85) or fragment, fusion polypeptides thereof, or a small moleculeTNF antagonist, e.g., TNF binding protein I or II (TBP-1 or TBP-II),nerelimonmab, infliximab, enteracept (EnbrelTM), adalimulab (Humira™),CDP-571, CDP-870, afelimomab, lenercept, and the like), antigen-bindingfragments thereof, and receptor molecules which bind specifically toTNF; compounds which prevent and/or inhibit TNF synthesis, TNF releaseor its action on target cells, such as thalidomide, tenidap,phosphodiesterase inhibitors (e.g, pentoxifylline and rolipram), A2badenosine receptor agonists and A2b adenosine receptor enhancers;compounds which prevent and/or inhibit TNF receptor signaling, such asmitogen activated protein (MAP) kinase inhibitors; compounds which blockand/or inhibit membrane TNF cleavage, such as metalloproteinaseinhibitors; compounds which block and/or inhibit TNF activity, such asangiotensin converting enzyme (ACE) inhibitors (e.g., captopril); andcompounds which block and/or inhibit TNF production and/or synthesis,such as MAP kinase inhibitors.

As used herein, a “tumor necrosis factor antibody,” “TNF antibody,”“TNFα antibody,” or fragment and the like decreases, blocks, inhibits,abrogates or interferes with TNFα activity in vitro, in situ and/orpreferably in vivo. For example, a suitable TNF human antibody of thepresent invention can bind TNFa and includes anti-TNF antibodies,antigen-binding fragments thereof, and specified mutants or domainsthereof that bind specifically to TNFa. A suitable TNF antibody orfragment can also decrease block, abrogate, interfere, prevent and/orinhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptorsignaling, membrane TNF cleavage, TNF activity, TNF production and/orsynthesis.

Chimeric antibody cA2 consists of the antigen binding variable region ofthe high-specificity neutralizing mouse anti-human TNFα IgG1 antibody,designated A2, and the constant regions of a human IgG1, kappaimmunoglobulin. The human IgG1 Fc region improves allogeneic antibodyeffector function, increases the circulating serum half-life anddecreases the immunogenicity of the antibody. The avidity and epitopespecificity of the chimeric antibody cA2 is derived from the variableregion of the murine antibody A2. In a particular embodiment, apreferred source for nucleic acids encoding the variable region of themurine antibody A2 is the A2 hybridoma cell line.

Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural andrecombinant human TNFα in a dose dependent manner. From binding assaysof chimeric antibody cA2 and recombinant human TNFα, the specificityconstant of chimeric antibody cA2 was calculated to be 1.04×10¹M⁻¹.Preferred methods for determining monoclonal antibody specificity andspecificity by competitive inhibition can be found in Harlow, et al.,antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988; Colligan et al., eds., Current Protocolsin Immunology, Greene Publishing Assoc. and Wiley Interscience, NewYork, (1992-2000); Kozbor et al., Immunol. Today, 4:72-79 (1983);Ausubel et al., eds. Current Protocols in Molecular Biology, WileyInterscience, New York (1987-2000); and Muller, Meth. Enzymol.,92:589-601 (1983), which references are entirely incorporated herein byreference.

In a particular embodiment, murine monoclonal antibody A2 is produced bya cell line designated c134A. Chimeric antibody cA2 is produced by acell line designated c168A.

Additional examples of monoclonal anti-TNF antibodies that can be usedin the present invention are described in the art (see, e.g., U.S. Pat.No. 5,231,024; Moller, A. et al., Cytokine 2(3):162-169 (1990); U.S.application Ser. No. 07/943,852 (filed Sep. 11, 1992); Rathj en et al.,International Publication No. WO 91/02078 (published Feb. 21, 1991);Rubin et al., EPO Patent Publication No. 0 218 868 (published Apr. 22,1987); Yone et al., EPO Patent Publication No. 0 288 088 (Oct. 26,1988); Liang, et al., Biochem. Biophys. Res. Comm. 137:847-854 (1986);Meager, et al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma6:359-369 (1987); Bringman, et al., Hybridoma 6:489-507 (1987); andHirai, et al., J. Immunol. Meth. 96:57-62 (1987), which references areentirely incorporated herein by reference).

Preferred TNF receptor molecules useful in the present invention arethose that bind TNFa with high specificity (see, e.g., Feldmann et al.,International Publication No. WO 92/07076 (published Apr. 30, 1992);Schall et al., Cell 61:361-370 (1990); and Loetscher et al., Cell61:351-359 (1990), which references are entirely incorporated herein byreference) and optionally possess low immunogenicity. In particular, the55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cell surface receptorsare useful in the present invention. Truncated forms of these receptors,comprising the extracellular domains (ECD) of the receptors orfunctional portions thereof (see, e.g., Corcoran et al., Eur. J.Biochem. 223:831-840 (1994)), are also useful in the present invention.Truncated forms of the TNF receptors, comprising the ECD, have beendetected in urine and serum as 30 kDa and 40 kDa TNFa inhibitory bindingproteins (Engelmann, H. et al., J. Biol. Chem. 265:1531-1536 (1990)).TNF receptor multimeric molecules and TNF immunoreceptor fusionmolecules, and derivatives and fragments or portions thereof, areadditional examples of TNF receptor molecules which are useful in themethods and compositions of the present invention. The TNF receptormolecules which can be used in the invention are characterized by theirability to treat patients for extended periods with good to excellentalleviation of symptoms and low toxicity. Low immunogenicity and/or highspecificity, as well as other undefined properties, can contribute tothe therapeutic results achieved.

TNF receptor multimeric molecules useful in the present inventioncomprise all or a functional portion of the ECD of two or more TNFreceptors linked via one or more polypeptide linkers or other nonpeptidelinkers, such as polyethylene glycol (PEG). The multimeric molecules canfurther comprise a signal peptide of a secreted protein to directexpression of the multimeric molecule. These multimeric molecules andmethods for their production have been described in U.S. applicationSer. No. 08/437,533 (filed May 9, 1995), the content of which isentirely incorporated herein by reference.

TNF immunoreceptor fusion molecules useful in the methods andcompositions of the present invention comprise at least one portion ofone or more immunoglobulin molecules and all or a functional portion ofone or more TNF receptors. These immunoreceptor fusion molecules can beassembled as monomers, or hetero- or homo-multimers. The immunoreceptorfusion molecules can also be monovalent or multivalent. An example ofsuch a TNF immunoreceptor fusion molecule is TNF receptor/IgG fusionprotein. TNF immunoreceptor fusion molecules and methods for theirproduction have been described in the art (Lesslauer et al., Eur. J.Immunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci.USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med. 174:1483-1489(1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994);Butler et al., Cytokine 6(6):616-623 (1994); Baker et al., Eur. J.Immunol. 24:2040-2048 (1994); Beutler et al., U.S. Pat. No. 5,447,851;and U.S. application Ser. No. 08/442,133 (filed May 16, 1995), each ofwhich references are entirely incorporated herein by reference). Methodsfor producing immunoreceptor fusion molecules can also be found in Caponet al., U.S. Pat. No. 5,116,964; Capon et al., U.S. Pat. No. 5,225,538;and Capon et al., Nature 337:525-531 (1989), which references areentirely incorporated herein by reference.

A functional equivalent, derivative, fragment or region of TNF receptormolecule refers to the portion of the TNF receptor molecule, or theportion of the TNF receptor molecule sequence which encodes TNF receptormolecule, that is of sufficient size and sequences to functionallyresemble TNF receptor molecules that can be used in the presentinvention (e.g., bind TNFα with high specificity and possess lowimmunogenicity). A functional equivalent of TNF receptor molecule alsoincludes modified TNF receptor molecules that functionally resemble TNFreceptor molecules that can be used in the present invention (e.g., bindTNFα with high specificity and possess low immunogenicity). For example,a functional equivalent of TNF receptor molecule can contain a “SILENT”codon or one or more amino acid substitutions, deletions or additions(e.g., substitution of one acidic amino acid for another acidic aminoacid; or substitution of one codon encoding the same or differenthydrophobic amino acid for another codon encoding a hydrophobic aminoacid). See Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, New York (1987-2003).

In some embodiments, the monoclonal antibody that inhibits TNF-a isselected from the group consisting of monoclonal rodent-humanantibodies, rodent antibodies, human antibodies or any portions thereof,having at least one antigen binding region of an immunoglobulin variableregion, which antibody binds TNF. Preferably, this monoclonal antibodyis selected from the group of compounds disclosed in U.S. Pat. No.6,277,969, the specification of which is incorporated by reference. Insome embodiments, the infliximab is delivered in a formulation having aninfliximab concentration of between about 30 mg/ml and about 60 mg/ml.

In some embodiments, the specific inhibitor of TNF-a is an inhibitor ofp38 MAP kinase, preferably, a small molecule inhibitor of p38 MAPkinase. The inhibition of p38 MAP kinase is believed to block productionof both TNF-a and I1-2, both of which are pro-inflammatory cytokines.The small molecule inhibitors of p38 MAP kinase are very specific &potent (˜nM). Without wishing to be tied to a theory, it is believedthat inhibition of p38 should not block TGF signaling nor TGF activity.It is further believed that p38 inhibitors may also block induction ofsome metalloproteinases, COX 2 and NO synthetase. It is further believedthat P38 inhibitors do not inhibit interleukins involved in immune cellproliferation such as IL-2.

In some embodiments, the HSCA is a specific antagonist of aninterleukin. Preferably, the target interleukin is selected from thegroup consisting IL-1, IL-2, IL-6 and IL-8, and IL-12. Preferredantagonists include but are not limited to Kineretg (recombinant IL1-RA, Amgen), IL1-Receptor Type 2 (Amgen) and IL-1 Trap (Regeneron).

The present inventors note that DDD involves the progressivedegeneration of a disc in which many factors are involved. In many ofthese instances, simply providing a single dose or even a regimen overthe space of a few days may not be sufficient to resolve the DDD. Forexample, if DDD were caused in part by mechanical instability in afunctional spinal unit, then simply providing a one-time therapy for thedisc cells will likely only delay the onset of the DDD. Therefore, thereis a need to provide a long-term drug therapy treatment of DDD that doesnot require multiple injections.

Because it is believed that the cytokines of interest both produce painand degrade the ECM when present within the nucleus pulposus, it isdesirable for the HSCA to remain within the nucleus pulposus as long aspossible in a pharmaceutically effective amount. The half-life of theHSCA within the nucleus pulposus will depend upon many factors,including the size of the HSCA and its charge. In general, the largerthe molecular weight of the HSCA, the more likely it is to remaincontained by the annulus fibrosus portion of the disc.

If the half-life of the HSCA is relatively short, then it would bedesirable for a relatively large dose of the HSCA to be administeredinto the disc. In this condition, quick depletion of the HSCA would notcause the HSCA to fall below therapeutically effective levels until anextended period.

Although a large dose of the HSCA would be desirable in such instances,it is also known that nociceptors present on the inner wall of theannulus fibrosus react to increased pressure and produce pain, and thatone avenue for increasing the pressure in the nucleus pulposus is toinject a critical volume of water. In some cases, the added amount couldbe as little as one cc by volume to produce pain. Accordingly, if adilute concentration of an HSCA is added to the nucleus pulposus toprovide a large dose, the resulting pressure increase caused by thisadded volume could be sufficient to cause acute pain.

For example, if it were determined that 100 mg of an HSCA was needed totherapeutically effect a nucleus pulposus, and that HSCA was provided inconcentrations of 30-60 mg/ml, then at least 1.5 ml of the HSCA wouldneed to be injected into the nucleus pulposus in order to provide thedesired therapeutic effect. However, when injecting volumes into thenucleus pulposus, it is desirable that the volume of drug delivered beno more than 1 ml, preferably no more than 0.5 ml, more preferablybetween 0.1 and 0.3 ml. When injected in these smaller quantities, it isbelieved the added volume will not cause an appreciable pressureincrease in the nucleus pulposus.

In contrast, Olmarker mixed 100 μl of a formulation comprising only 1.11mg/ml of a monoclonal antibody into 40 mg of an extracted nucleuspulposus.

Accordingly, in some embodiments, the concentration of the TNF-aantagonist in the administered drug is at least 100 mg/ml. When 100 mgof the HSCA is needed to produce the desired therapeutic result, no morethan 1 ml of the drug need be injected. Preferably, the concentration ofthe TNF-a antagonist in the administered drug is at least 200 mg/ml. Inthis condition, no more than 0.5 ml of the drug need be injected.Preferably, the concentration of the TNF-a antagonist in theadministered drug is at least 500 mg/ml. In this condition, between 0.03and 0.3 ml of the drug need be injected.

In some embodiments, the HSCA is provided in a sustained release device.The sustained release device is adapted to remain within the disc for aprolonged period and slowly release the HSCA contained therein to thesurrounding environment. This mode of delivery allows an HSCA to remainin therapeutically effective amounts within the disc for a prolongedperiod.

In some embodiments, the HSCA is predominantly released from thesustained delivery device by its diffusion through the sustaineddelivery device (preferably, though a polymer). In others, the HSCA ispredominantly released from the sustained delivery device by thebiodegradation of the sustained delivery device (preferably,biodegradation of a polymer).

Preferably, the sustained release device comprises a bioresorbablematerial whose gradual erosion causes the gradual release of the HSCA tothe disc environment. In some embodiments, the sustained release devicecomprises a bioresorbable polymer. Preferably, the bioresorbable polymerhas a half-life of at least one month, more preferably at least twomonths, more preferably at least 6 months.

In some embodiments, the sustained release device provides controlledrelease. In others, it provides continuous release. In others, itprovides intermittent release. In others, the sustained release devicecomprises a biosensor.

In some embodiments, the sustained delivery device comprises bioerodablemacrospheres. The HSCA is preferably contained in a gelatin (or water orother solvent) within the capsule, and is released to the discenvironment when the outer shell has been eroded. The device can includea plurality of capsules having outer shells of varying thickness, sothat the sequential breakdown of the outer shells provides periodicrelease of the HSCA.

In some embodiments, the sustained delivery device comprises aninflammatory-responsive delivery system, preferably comprisingbioerodable microspheres that are eroded by invading macrophages. Thistechnology provides a high correspondence between physiologicinflammation of disc environment and the release of the HSCAs into thatenvironment. Preferably, the technology disclosed in Brown et al.,Arthritis. Rheum. 1998 Dec.; 41(12) pp., 2185-95 is selected.

In some embodiments, the sustained delivery device comprises the devicesdisclosed in U.S. Pat. No. 5,728,396 (“Peery”), the specification ofwhich is incorporated by reference in its entirety.

In some embodiments, the sustained delivery device comprises a plurality(preferably at least one hundred) of water-containing chambers, eachchamber containing the HSCA. Each chamber is defined by bilayer lipidmembranes comprising synthetic duplicates of naturally occurring lipids.The release of the drug can be controlled by varying at least one of theaqueous excipients, the lipid components, and the manufacturingparameters. Preferably, the formulation comprises no more than 10%lipid. In some embodiments, the Depofoam™ technology of Skyepharma PLC(located in London, United Kingdom) is selected.

In some embodiments, the sustained delivery device comprises a deliverysystem disclosed in U.S. Pat. No. 5,270,300 (“Hunziker”), thespecification of which is incorporated by reference in its entirety.

In some embodiments, the sustained delivery device comprises theco-polymer poly-DL-lactide-co-glycolide (PLG). Preferably, theformulation is manufactured by combining the HSCA, the co-polymer and asolvent to form a droplet, and then evaporating the solvent to form amicrosphere. The plurality of microspheres are then combined in abiocompatible diluent. Preferably, the HSCA is released from theco-polymer by its diffusion therethrough and by the biodegradation ofthe co-polymer. In some embodiments hereof, the ProLease™ technology ofAlkermes (located in Cambridge, Mass.) is selected.

Hydrogels can also be used to deliver the HSCA is a time-release mannerto the disc environment. A “hydrogel” is a substance formed when anorganic polymer (natural or synthetic) is set or solidified to create athree- dimensional open-lattice structure that entraps molecules ofwater or other solution to form a gel. The solidification can occur,e.g., by aggregation, coagulation, hydrophobic interactions, orcross-linking. The hydrogels employed in this invention rapidly solidifyto keep the HSCA at the application site, thereby eliminating undesiredmigration from the disc. The hydrogels are also biocompatible, e.g., nottoxic, to cells suspended in the hydrogel.

A “hydrogel-HSCA composition” is a suspension of a hydrogel containingdesired HSCA. The hydrogel-HSCA composition forms a uniform distributionof HSCA with a well-defined and precisely controllable density.Moreover, the hydrogel can support very large densities of HSCA. Inaddition, the hydrogel allows diffusion of nutrients and waste productsto, and away from, the HSCA, which promotes tissue growth.

Hydrogels suitable for use in the present invention includewater-containing gels, i.e., polymers characterized by hydrophilicityand insolubility in water. See, for instance, “Hydrogels”, pages 458-459in Concise Encyclopedia of Polymer Science and Engineering, Eds. Mark etal., Wiley and Sons, 1990, the disclosure of which is incorporatedherein by reference. Although their use is optional in the presentinvention, the inclusion of hydrogels is highly preferred since theytend to contribute a number of desirable qualities. By virtue of theirhydrophilic, water-containing nature, hydrogels can:

a) house viable cells, such as mesenchymal stems cells, and

b) assist with load bearing capabilities of the disc.

In a preferred embodiment, the hydrogel is a fine, powdery synthetichydrogel. Suitable hydrogels exhibit an optimal combination of suchproperties as compatibility with the matrix polymer of choice, andbiocompatability. The hydrogel can include any of the following:polysaccharides, proteins, polyphosphazenes,poly(oxyethylene)-poly(oxypropylene) block polymers,poly(oxyethylene)-poly(oxypropylene) block polymers of ethylene diamine,poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acidand methacrylic acid, poly(vinyl acetate), and sulfonated polymers.

In general, these polymers are at least partially soluble in aqueoussolutions, e.g., water, or aqueous alcohol solutions that have chargedside groups, or a monovalent ionic salt thereof. There are many examplesof polymers with acidic side groups that can be reacted with cations,e.g., poly(phosphazenes), poly(acrylic acids), and poly(methacrylicacids). Examples of acidic groups include carboxylic acid groups,sulfonic acid groups, and halogenated (preferably fluorinated) alcoholgroups. Examples of polymers with basic side groups that can react withanions are poly(vinyl amines), poly(vinyl pyridine), and poly(vinylimidazole).

In some embodiments, the sustained delivery device includes a polymerselected from the group consisting of PLA, PGA, PCL, and mixturesthereof.

If the half-life of the HSCA within the disc is relatively long, then itmay be assumed that a relatively small dose of the HSCA can beadministered into the disc. In this condition, the slow depletion of theHSCA would not cause the HSCA to fall below therapeutically effectivelevels in the disc until an extended period of time has elapsed.

In some embodiments in which HSCAs have long half-lives within the disc,the dose administered can be very small.

For example, if it is believed that an HSCA is effective when present inthe range of 1-10 mg/kg or 1-10 ppm (as is believed to be the case forthe TNF antagonist Remicade™), and since a typical nucleus pulposus of adisc has a volume of about 3 ml (or 3 cc, or 3 g), then only about 3-30ug of the HSCA need be administered to the disc in order to provide along lasting effective amount of the drug. As a point of reference,Tobinick discloses that at least 1 mg of cytokine antagonist should beadministered perispinally in order to cure back pain. Similarly,Olmarker mixed 100 ml of a formulation comprising 1.11 mg/ml of amonoclonal antibody into 40 mg of an extracted nucleus pulposus, therebyproducing a monoclonal antibody concentration of about 3 parts perthousand. The smaller amounts available by this route reduce the chancesof deleterious side effects of the HSCA.

For example, suppose a clinician administered 0.3 ml of 60 mg/mlinfliximab into a 2.7 cc disc, thereby producing a infliximabconcentration in the disc of about 6 mg/ml, or 6 parts per thousand.Without wishing to be tied to a theory, if infliximab has the samehalf-life within a nucleus pulposus as it does when administeredsystemically (i.e., about 1 week), then the concentration of infliximabwould remain above about 10 ppm for about 9 weeks. Therefore, if anotherdose were needed, the clinician would only need to provide the seconddose after about two months.

Therefore, in some embodiments, the HSCA is provided in a dose of lessthan 1 mg, preferably, less than 0.5 mg, more preferably, less than 0.1mg, more preferably less than 0.01 mg. The smaller amounts available bythis route reduce the chances of deleterious side effects of the HSCA.Preferably, the HSCA provided in these smaller amounts is a TNFantagonist, more preferably is infliximab.

In preferred embodiments, the formulation of the present invention isadministered directly into the disc through the outer wall of theannulus fibrosus. More preferably, the direct administration includesdepositing the HSCA in the nucleus pulposus portion of the disc. In thiscondition, the fibrous nature of the annulus fibrosus that surrounds thenucleus pulposus will help keep the HSCA contained within the disc.

Preferably, the formulation of the present invention is injected intothe disc through a small bore needle. More preferably, the needle has abore of 22 gauge or less, so that the possibilities of producing aherniation are mitigated. More preferably, the needle has a bore of 24gauge or less, so that the possibilities of producing a herniation areeven further mitigated.

If the volume of the direction injection of the formulation issufficiently high so as to cause a concern of overpressurizing thenucleus pulposus, then it is preferred that at least a portion of thenucleus pulposus be removed prior to direct injection. Preferably, thevolume of removed nucleus pulposus is substantially similar to thevolume of the formulation to be injected. More preferably, the volume ofremoved nucleus pulposus is within 80-120% of the volume of theformulation to be injected. In addition, this procedure has the addedbenefit of at least partially removing some degenerated disc from thepatient.

In other embodiments, the formulation is delivered into the disc spacethrough the endplate of an opposing vertebral body. This avenueeliminates the need to puncture the annulus fibrosus, and so eliminatesthe possibility of herniation.

Although the cytokine antagonists may therapeutically treat the disc bybinding the target cytokine, and thereby reducing pain and arrestingdegradation of the ECM, it is believed that at least some of theseantagonists do not help repair the damage done by the cytokine to theECM.

Therefore, there may be a need to provide a therapy that also helpsrepair the ECM.

In accordance with the present invention, there is provided a method oftreating degenerative disc disease in an intervertebral disc having anucleus pulposus, comprising the steps of:

-   -   a) administering a highly specific cytokine antagonist into a        degenerating disc; and    -   b) administering a second therapeutic agent in an amount        effective to at least partially repair the disc.

In accordance with one aspect of the invention, both the HSCA and secondtherapeutic agent are locally administered into the disc. Because theHSCA is specific, it does not interfere with the locally administeredsecond therapeutic agent, and so each agent may independently work toprovide therapy to the diseased disc.

In some embodiments, the HSCA and second therapeutic agent areadministered simultaneously. In others, the HSCA is administered first.In still others, the second therapeutic agent is administered first.

Other compounds which may be added to the disc include, but are notlimited to: vitamins and other nutritional supplements; hormones;glycoproteins; fibronectin; peptides and proteins; carbohydrates (bothsimple and/or complex); proteoglycans; oligonucleotides (sense and/orantisense DNA and/or RNA); BMPs; antibodies (for example, to infectiousagents, tumors, drugs or hormones); and gene therapy reagents.Genetically altered cells and/or other cells may also be included in thematrix of this invention. If desired, substances such as pain killersand narcotics may also be admixed with a polymer for delivery andrelease to the disc space.

Preferably, healthy cells are introduced into the disc that have thecapability of at least partially repairing any damage done to the discduring the degenerative process. In some embodiments, these cells areintroduced into the nucleus pulposus and ultimately produce newextracellular matrix for the nucleus pulposus. In others, these cellsare introduced into the annulus fibrosus and produce new extracellularmatrix for the annulus fibrosus.

In some embodiments, these cells are obtained from another humanindividual (allograft), while in others, the cells are obtained from thesame individual (autograft). In some embodiments, the cells are takenfrom an intervertebral disc (and can be either nucleus pulposus cells orannulus fibrosus cells), while in others, the cells are taken from anon-disc tissue (and may be mesenchymal stem cells). In others,autograft chondrocytes (such as from the knee, hip, shoulder, finger orear) may be used.

Preferably, when viable cells are selected as the second therapeuticsubstance, the viable cells comprise mesenchymal stem cells (MSCs). MSCsprovide a special advantage for administration into a degenerating discbecause it is believed that they can more readily survive the relativelyharsh environment present in the degenerating disc; that they have adesirable level of plasticity; and that they have the ability toproliferate and differentiate into the desired cells.

In some embodiments, the mesenchymal stems cells are obtained from bonemarrow, preferably autologous bone marrow. In others, the mesenchymalstems cells are obtained from adipose tissue, preferably autologousadipose tissue.

In some embodiments, the mesenchymal stem cells injected into the discare provided in an unconcentrated form. In others, they are provided ina concentrated form. When provided in concentrated form, they arepreferably uncultured. Uncultured, concentrated MSCs can be readilyobtained by centrifugation, filtration, or immuno-absorption. Whenfiltration is selected, the methods disclosed in U.S. Pat. No. 6,049,026(“Muschler”), the specification of which is incorporated by reference inits entirety, are preferably used. In some preferred embodiments, thematrix used to filter and concentrate the MSCs is also administered intothe nucleus pulposus. If this matrix has suitable mechanical properties,it can be used to restore the height of the disc space that was lostduring the degradation process.

As used herein, the term “growth factors” encompasses any cellularproduct that modulates the growth or differentiation of other cells,particularly connective tissue progenitor cells. The growth factors thatmay be used in accordance with the present invention include, but arenot limited to, members of the fibroblast growth factor family,including acidic and basic fibroblast growth factor (FGF-1 and -2) andFGF-4, members of the platelet-derived growth factor (PDGF) family,including PDGF-AB, PDGF-BB and PDGF-AA; EGFs, members of theinsulin-like growth factor (IGF) family, including IGF-I and -II; theTGF-β superfamily, including TGF-β1, 2 and 3 (including MP-52),osteoid-inducing factor (OIF), angiogenin(s), endothelins, hepatocytegrowth factor and keratinocyte growth factor; members of the bonemorphogenetic proteins (BMP's) BMP-1, (BMP-3); BMP-2; OP-1; BMP-2A, -2B,and -7, BMP-14 ; HBGF-1 and -2; growth differentiation factors (GDF's),members of the hedgehog family of proteins, including indian, sonic anddesert hedgehog; ADMP-1; members of the interleukin (IL) family,including IL-1 thru -6; GDF-5 and members of the colony-stimulatingfactor (CSF) family, including CSF-1, G-CSF, and GM-CSF; and isoformsthereof.

In some embodiments, the growth factor is selected from the groupconsisting of TGF-B, bFGF, and IGF-1. These growth factors are believedto promote regeneration of the nucleus pulposus. Preferably, the growthfactor is TGF-B. More preferably, TGF-B is administered in an amount ofbetween 10 ng/ml and 5000 ng/ml, more preferably between 50 ng/ml and500 ng/ml, more preferably between 100 ng/ml and 300 ng/ml.

In some embodiments, platelet concentrate is provided as the secondtherapeutic agent. Preferably, the growth factors released by theplatelets are present in an amount at least two-fold (more preferably,four-fold) greater than the amount found in the blood from which theplatelets were taken. More preferably, the platelet concentrate isautologous. In some embodiments, the platelet concentrate is plateletrich plasma (PRP). PRP is advantageous because it contains growthfactors that can restimulate the growth of the ECM, and because itsfibrin matrix provides a suitable scaffold for new tissue growth.

Since it is known that many pro-inflammatory proteins play a role indisc degeneration, and that the antagonists of the present invention arehighly specific, it is further believed that injecting at least two ofthe highly specific antagonists of the present invention directly intothe disc would be advantageous.

Therefore, in accordance with the present invention, there is provided amethod of treating degenerative disc disease in an intervertebral dischaving a nucleus pulposus, comprising the steps of:

-   -   a) administering a formulation comprising at least two highly        specific antagonists of pro-inflammatory cytokines selected from        the group consisting of TNF-α, an interleukin (preferably, IL-1,        I1-6 and IL-8), FAS, an FAS ligand, and IFN-gamma.

Preferably, at least one of the substances is an antagonist of TNF-α.Preferably, the other substance is an antagonist of an interleukin.

In some embodiments, the formulation comprises a suitable biocompatiblecarrier such as saline. In some embodiments, the carrier is selectedfrom the carriers disclosed in U.S. Pat. No. 6,277,969 (“Le”), thespecification of which is incorporated by reference in its entirety.

Also in accordance with the present invention, there is provided aformulation for treating degenerative disc disease, comprising:

-   -   a) a high specificity cytokine antagonist, and    -   b) a second therapeutic agent selected from the group consisting        of:        -   i) a growth factor, and        -   ii) viable cells.

In some embodiments of this formulation, the high specificity cytokineantagonist is selected from the group consisting of antagonists of TNFand antagonists of an interleukin.

Because the causes of low back pain may be myriad, and because of thesignificant cost of many of these specialized HSCAs, it would be usefulfor the clinician to first perform a diagnostic test in order to confirmthat the targeted disc in fact possesses high levels of the targetedcytokine prior to providing the injection.

In one embodiment, the diagnostic test comprises a non-invasivediagnostic test comprising using an MRI.

Preferably, the clinician would first perform a discogram in order toidentify which disc or discs are responsible for the patient's low backpain. Next, the clinician would perform an invasive or non-invasive testupon the targeted disc in order to confirm the presence of or quantifythe level of the pro-inflammatory cytokine.

In one embodiment, the diagnostic test comprises an invasive test inwhich a portion of the disc is removed and analyzed. In someembodiments, the clinician removes a portion of the nucleus pulposus. Inothers, the clinician removes a portion of the annulus fibrosus.Preferably, the removed material is a portion of the nucleus pulposus.The presence of pro-inflammatory cytokines in the removed material maydetected by procedures including but not limited to electrophoresis, oran enzyme-linked immunoabsorbent assay (as per Burke, Br. JBJS, 84-B(2),2002).

In some embodiments, the diagnostic methods disclosed in U.S. Pat. No.6,277,969 (“Le”), the specification of which is incorporated byreference in its entirety, are selected. In these methods, highspecificity anti-TNF-α compounds are used as diagnostic tools fordetecting TNF-alpha in the patient known or suspected to have a highlevel of TNF-alpha.

After determining the levels of the different pro-inflammatory cytokinesin the degenerating disc, the clinician will preferably proceed tocompare these diagnosed levels against pre-determined levels of thepro-inflammatory cytokines. If the diagnosed level of thepro-inflammatory cytokine exceeds the pre-determined level, then theclinician may conclude that these higher levels are causing unwantedinflammatory action and proceed to directly inject a specific HSCA intothe disc capable of inhibiting the targeted protein.

In some embodiments, the predetermined level for an interleukin is atleast 100 pg/ml. In some embodiments, the predetermined level for IL-6is at least 250 pg/ml. In some embodiments, the predetermined level forIL-8 is at least 500 pg/ml. In some embodiments, the predetermined levelfor PGE2 is at least 1000 pg/ml. In some embodiments, the predeterminedlevel for TNF-α is at least 500 pg/ml. In others, the predeterminedlevel for TNF-α is at least 20 pg/ml, more preferably at least 30 pg/ml,more preferably at least 50 pg/ml, more preferably at least 1 ng/ml. Inothers, the predetermined level for TNF-α is at least 1 ng/disc.

It would also be useful to be able to determine whether directlyadministering the therapeutic substances of the present invention was infact efficacious. Accordingly, one can measure the level of cytokineremaining in the disc after administration.

It is further believed that the present invention can also be used toprevent degeneration of an intervertebral disc in a human individual,namely, by following a procedure comprising the steps of:

-   -   a) determining a genetic profile of the individual,    -   b) comparing the profile of the individual against a        pre-determined genetic profile level of at-risk humans,    -   c) determining that the individual is at at-risk patient, and    -   d) injecting an antagonist of the pro-inflammatory protein into        a disc of the individual.

EXAMPLE I

This non-limiting prophetic example describes how to transdiscallyadminister a formulation comprising a HSCA and saline into a nucleuspulposus of a degenerating disc.

First, the clinician uses a diagnostic test to verify that a particulardisc within a patient has high levels of a particular pro-inflammatorycytokine.

Next, the clinician provides a local anesthetic (such as 5 ml lidocaine)to the region dorsal of the disc of concern to reduce subcutaneous pain.

Next, the clinician punctures the skin of the patient dorsal the disc ofconcern with a relatively large (e.g., 18-19 gauge) needle having astylet therein, and advances the needle through subcutaneous fat anddorsal sacrolumbar ligament and muscles to the outer edge of theintervertebral disc.

Next, the stylet is removed from the needle.

Next, the clinician receives a syringe having a smaller gauge needleadapted to fit within the larger gauge needle. This needle is typicallya 22 or 24 gauge needle. The barrel of the syringe contains theformulation of the present invention.

The formulation contains infliximab, and has an infliximab concentrationof between about 30 mg/ml and about 60 mg/ml.

Next, the physician advances the smaller needle co-axially through thelarger needle and past the distal end of the larger needle, therebypuncturing the annulus fibrosus. The smaller needle is then furtheradvanced into the center of the nucleus pulposus. Finally, the cliniciandepresses the plunger of the syringe, thereby injecting between about0.1 and 1 ml of the formulation into the nucleus pulposus.

EXAMPLE II

This non-limiting prophetic example is substantially similar to that ofExample I, except that the formulation comprises a sustained releasedevice comprising the co-polymer poly-DL-lactide-co-glycolide (PLG). Theformulation contains infliximab as the antagonist, and has an infliximabconcentration of between about 30 mg/ml and about 60 mg/ml.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-20. (canceled)
 21. A method of treating a patient having adegenerating disc, comprising the step of: a) injecting into the disc aformulation comprising: i) cultured, allograft intervertebral disccells; and ii) fibrinogen; and b) forming within the disc a fibrinmatrix derived from the fibrinogen that provides a scaffold for newtissue growth.
 22. The method of claim 21, wherein the formulationfurther comprises iii) thrombin.